This invention relates to methods for high throughput screening of candidate drug compounds, which finds particular use in the rapid and highly sensitive determination of drug bioactivity and drug target identification.
Traditionally the pharmaceutical industry has relied on two principal methods for drug discovery: 1) in vitro, cell-free biochemical assays; and 2) cell-based assays. In in vitro, cell-free biochemical assays, a massive library of compounds is screened against a given target. Biochemical assays identify compounds of interest by detecting the ability of the compound to alter activity of the target (e.g., by decreasing or increasing an enzymatic activity). The rapidity and efficiency of such screening methods have improved with the advent of automated techniques and advances in computer technology, thus facilitating discovery of important drugs (Palmer, 1996 Nature Biotech. 14:513-515).
However, the effectiveness of this high throughput approach to drug screening depends on the ability to design bioassays to test the activity of the target in the screen. The choice of target is then limited, in part, by the efficacy of designing a suitable bioassay amenable to automation. This also requires significant a priori knowledge of the target. In addition, initial target selection is biased since investigators are often forced to select possible targets based only upon a combination of hearsay and empirical experience. Once a compound having a desired activity has been discovered using a biochemical in vitro cell-free assay, several caveats remain including whether the compound will interact with the target in vivo as it did in the cell-free in vitro assay, whether the compound will enter the cell to reach the target, whether it will be stable in vivo, and whether the compound will specifically affect the desired target without affecting non-target gene products, either specifically or nonspecifically. In addition, this screening method makes it difficult to study natural broths or drug mixtures where drug concentrations may be too low to detect any alteration in target activity.
In a second drug discovery method, the compounds are screened for a desired effect in a cell-based assay. Unlike in vitro biochemical assays, cell-based assays are based upon the ability of a compound(s) to affect some function or aspect of an entire organism and will identify compounds that have biologically significant effects. Conventional cell-based assays, however, also have limitations. For example, suitable in vivo assays must be designed, limiting the choice of targets. Cell-based assays can result in identification of compounds that non-specifically affect the cells. For example, investigators can use cell-based assays to identify compounds that generally affect cell growth, but since growth inhibitors may affect any of a variety of cell structures or enzymes, the investigators cannot immediately and directly identify the specific target of the inhibitory compound. In these cases, the drug is either used without knowledge of the target, or more likely, is found to be of limited use due to nonspecific cytotoxic effects.
A direct approach for identification of drug targets is based on the notion that dramatically increasing dosage of the target gene (e.g., through the use of multicopy plasmids), and thus overexpressing the target gene product, will confer resistance to certain drugs. Thus, target gene products can be identified by constructing a library of cells, each cell carrying a multiple copy plasmid expressing a different candidate target gene, and growing the library of cells in the presence of drug to select for those recombinant cells that, by virtue of increased dosage of the target gene, exhibit increased resistance to the drug. Evidence that overexpression of a gene can alter its sensitivity to a drug has been demonstrated (see, e.g. Barnes et al. (1984) Mol Cell. Biol. 4:2381-88; Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750-6754; Rine (1991) Meth. Enzymol. 194:239-251).
Although gene overexpression using high copy number plasmids is a powerful technique for identifying gene products of interest, this approach has certain limitations. Gene product overexpression can itself be lethal to a host cell or can significantly alter the cell""s usual biological pathways and processes (Liu, H. et al. (1992) Genetics 132:665-673). Moreover, the copy number of the plasmid containing the gene of interest is not easily controlled or predictable (Rine, J. (1991), supra). Furthermore, growth under selective conditions, especially for long periods of time (e.g., days to weeks), encourages selection of mutant cells that may be altered in expression of gene products other than the gene product carried on the plasmid (e.g. second site suppressors). Thus, the target gene identified using this selection process does not always identify the true target of the drug. Finally, such gene overexpression assays can be time-consuming, as the method requires additional screening of drug-resistant clones to identify targets.
Another approach, described by Giaever, G. et al. (1999) Nature Genet. 21(3):278-283, takes advantage of the observation that the copy number of a gene encoding a drug target directly and sensitively determines the host cell""s sensitivity to the drug to the degree that altering the target gene copy number by only one copy (e.g., from two copies to one copy) elicited a detectable phenotypic change (e.g., a change in growth rate or fitness of the strain) in the presence of the drug. Thus, drugs and their target gene products can be identified by, for example, identification of heterozygous deletion strains that exhibit a slower growth rate in the presence of drug relative to wildtype. Furthermore, this genomic approach is parallel and quantitative: all strains, and therefore all targets, are simultaneously measured for drug sensitivities. While this approach is powerful, it requires use of currently costly DNA microarrays as well as upfront determination of drug activities, which, though straightforward, can be time consuming.
Ideally, one would like a highly sensitive method for high-throughput screening of thousands of candidate agents simultaneously, either separately, or in cocktails, which would allow both the determination of whether a candidate agent is active as a drug, and what the target of the drug might be. In short, there is a need in the field for a simple assay that provides information about the drug activity of candidate compounds using a rapid, sensitive and inexpensive global indicator of such activity that can be easily detected. In addition, once this drug activity is determined, it can be used to determination the drug target of the compound. The present invention addresses this problem.
Targeted selection of recombinant yeast clones encoding drug resistance by dramatically increasing gene dosage is described in Rine, J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750-6754.; Rine, J. (1991) Methods Enzymol. 194:239-251.
Genomic profiling of drug sensitivities via induced haploinsufficiency is described by Giaever, G. et al. (1999) Nature Genet. 21(3):278-283.
Analysis of yeast deletion mutants using a molecular bar-coding strategy is described in Shoemaker, D. D. et al. (1996) Nature Genet. 14:450-456.
Drug target validation and identification of xe2x80x9coff-targetxe2x80x9d secondary drug effects using DNA microarrays is described by Marton, M. J. et al. (1998) Nature Med. 4:1293-1301.
Functional Classification of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis in Winzeler, E. et al. (1999) Science 285:901-906, see also the Stanford University yeast deletion project worldwide website at stanford.edu/group/yeast_deletionxe2x80x943.html.
The complete sequence of the genome of S. cerevisiae is available from the Stanford University Saccharomyces genome worldwide website at stanford.edu/Saccharomyces, and is discussed in Goffeau, A et al. (1996) Science 274:563-567.
The invention features methods of high throughput screening of candidate drug agents and rapid identification of drug targets by examining induction of the stress response in a host cell. In general, induction of the stress response in wildtype host cells indicates that a candidate agent has activity on the cell. Induction of a relatively lower or undetectable stress response in a host cell comprising an decrease in gene product dosage of a drug target relative to the wildtype state) indicates that the host cell is drug-sensitive and is altered in a gene product that may encode the target because in this case the cell may be too sick to illicit a xe2x80x9chealthyxe2x80x9d stress response due to the further inhibition of the gene product in the presence of drug. Similarly, induction of a relatively lower or undetectable stress response in a host cell either resistant to drug or carrying an increase in drug copy number (with respect to wildtype) indicates that the host cell is drug resistant relative to wildtype. The induction of the stress response can be assessed using reporter constructs for selected stress response genes, thus facilitating high throughput screening.
One advantage of the invention is that candidate agents having activity as drugs can be rapidly identified by quantitative or qualitative detection of expression of a single gene (e.g., rather than examining the relative expression levels of multiple genes) in an inexpensive and highly sensitive assay.
Another advantage of the invention is that the methods of the invention exploit the stress response, which is normally regarded as unavoidable xe2x80x9cbackground noise,xe2x80x9d to rapidly identify candidate agents having drug activity and, where desired, identify the target gene products of the drug activity. Importantly, this stress response often clouds interpretation of microarray expression experiments, forcing the sensitivity to be lowered. The present invention exploits the robust stress response of cells to provide a sensitive metric of drug effectiveness.
Another advantage of the invention is that the host cells having varying copy numbers of the target sequences (or which express the target gene product-encoding sequence at varying levels) are easy to construct and collections can be custom designed to screen for drugs that inhibit specific cellular processes and targets. To date over 80% of the complete heterozygous deletion collection have been constructed. Yeast will not only be useful for the identification of antifungals but will also provide a good model system for identification of cellular processes and pathways that yeast share with humans.
Yet another advantage of the invention is that it does not require overexpression of the candidate target gene product sequence, which can be lethal. Moreover, the effect of the drug and identification of deletion strains that carry a deletion for a target gene product can be identified without the need to assess growth rate, i.e., only a snapshot of stress response gene activity is necessary. Thus the test can be conducted very quickly using induced RNA in the presence of stress. For example, Taqman(trademark) assays can be used to detect the response in only a few minutes.
Still another advantage is that the invention can identify multiple targets of a drug. For example, the method of the invention can lead to identification of several different drug-sensitive host cells, each of which contains deletions of a gene that may encode a drug target, thus identifying any multiple targets if they exist.
Still another advantage is that the invention takes advantage of available genomic information. For example, if the complete or partial sequence of a potential target gene(s) is known, then one can readily construct host cells for use in the invention, even if the function of the gene is unknown. In addition, the host cells need not be heterozygotes for a possible target gene. Instead the host cells could carry an increase in gene dosage. In this embodiment, the expectation is that the stress response would be reduced when compared to that of an unaltered host cell.
Another advantage of the invention is that information about the function of unknown genes can be readily attained in an in vivo system. For example, identifying strains that are sensitive or resistant to a known drug may reveal genes not otherwise known to be involved in the same pathway or parallel pathways. Furthermore, large-scale analysis of drug experiments will undoubtedly reveal clustering of experiments with respect to drug classes and, in addition, with respect to groups of genes that may define functional categories, thus aiding in elucidation of pathways. Indeed, each experiment performed in this manner reiterates the previous experiments, and, in this way, will allow the development of a database of stress response profiles. This database in turn will allow the identification of the xe2x80x9cbestxe2x80x9d reporter genes.
The method of the invention is also advantageous in that it allows screening of many potential drug targets simultaneously (e.g. gene products involved in any cellular process such as DNA synthesis, assembly and function of the mitotic apparatus, sterol biosynthesis, cell wall biosynthesis, etc.), rather than testing such targets individually using conventional methods. For example, all possible targets can be tested using a collection of strains having deletions representative of the entire host cell""s genome (e.g., using yeast as a host cell). Such a collection of yeast strains is presently under construction, and is over 80% complete. Moreover, the invention allows for the identification of drug targets for known and new compounds alike, as well as the identification of new drugs that inhibit the same target as a known drug. Furthermore, the rapid nature of the method allows for rapid analysis of several different concentrations of the drug or drug candidates.
Another advantage of the invention is that once a target gene product(s) of a known drug is identified, or a target of interest is chosen, the method can be used to identify other drugs that bind that same target gene product(s) and/or affect the same pathway, thereby facilitating development of drugs that have greater specificity for a target gene product and/or target pathway, elicit fewer side effects, and are safe and effective, thus speeding the drug development process.
The method of the invention is also advantageous in that it is extremely sensitive, thus allowing detection of bioactive compounds at very low concentrations of the candidate compound. The invention thus allows discovery of drugs that would previously go undetected, and requires only a very small amount of candidate compound for screening. For example, assays for detection of drugs can be carried out with 100 host cells and possibly with as few as 10 host cells.
Yet another advantage of the invention is that detection of expression levels of a stress response gene (e.g., through use of a reporter gene construct or through direct measurement of induced RNA) is at least an order of magnitude more sensitive relative to conventional drug inhibition assays, e.g., the method of the invention can be used in place of a halo assay for drugs that do not normally result in a detectable zone of inhibition. For example, host cells could be provided in microtiter plates, drug or candidate drug added to a well(s) of the microtiter plate, and a detectable marker associated with expression of a stress response gene(s) detected. The invention thus provides for a quick, inexpensive, simple assay. These assays require only small volumes of compounds and low numbers of host cells (e.g., as few as 100 cells/well or less), and are 1,000 to 10,000 times more sensitive than, for example, halo assays on plates or conventional liquid culture assays.
The invention is also advantageous in that it can facilitate identification of multiple drugs in a mixture or complex mixture of natural origin, even if the drugs within the mixture have different targets or are present in very low concentrations. Furthermore, the method allows identification of the targets of the drugs within these mixtures.
In addition, it should be noted that the invention also provides valuable information in eliminating those compounds that do not exhibit drug activity in a quick, inexpensive assay. In this aspect, the assay avoids investment of time and money into characterization of compounds that will not be good candidates for further drug development.
The method of the invention is also advantageous in that it is readily adapted to high-throughput automation, is highly parallel and quantitative, and is rapid (i.e., assays can be carried out in a few hours or in some cases in a manner of minutes). Moreover, because the assays do not require long term growth (i.e., not more than a few hours) the probability of selection for secondary mutations that might mask the effect of the true target gene is minimized.
Still another advantage of the invention is that it is relatively inexpensive to screen multiple drugs since, for example, collections of strains representing a large number of targets can be screened simultaneously in small culture volumes (100 xcexcl or less), thus requiring only small amounts of drug. In addition, the equipment and supplies needed for this type of assay is minimal; i.e., requiring only off the shelf robotics, microtiter plates, media, and a fluorescent plate reader.
Another advantage of the invention is that it can be readily adapted for use with a wide variety of host cells (e.g., haploid or diploid organisms; bacterial, yeast, or mammalian cells) and with a wide variety of candidate target gene product sequences.